The present invention relates to methods for the treatment of liposarcomas. In another aspect, the present invention relates to compounds and compositions which are useful for carrying out the above-referenced methods.
Liposarcoma is the most common soft tissue malignancy in adults, accounting for at least 20% of all sarcomas in this age group. Multiple histologic subtypes of liposarcoma are recognized, including well differentiated, de-differentiated, myxoid, round cell and pleimorphic. The histologic subtype is predictive of both the clinical course of the disease and the ultimate prognosis.
Localized disease is treated primarily with surgery, often in combination with radiotherapy. Metastatic liposarcoma is associated with an extremely poor prognosis, with average five year survival ranging from 70% to 25%, depending on the type of tumor. Unfortunately, conventional chemotherapy for metastatic liposarcoma leads to complete response in only about 10% of cases. Thus, for most patients, conventional chemotherapy is largely palliative.
Induction of terminal differentiation represents a promising alternative to conventional chemotherapy for certain malignancies. For example, the retinoic acid receptor alpha (RARxcex1), which plays an important role in the differentiation and malignant transformation of cells of myelocytic lineage, has been used as a target for intervention in acute promyelocytic leukemia. Indeed, differentiation therapy with all-trans retinoic acid has become the standard of care for this disease. In view of this success, it has been speculated that nuclear receptors that regulate growth and differentiation of other cell types may also represent potential targets for differentiation therapy.
Recent years have seen important advances in the understanding of the molecular basis of adipocyte differentiation. Central to this process is the induction of the adipocyte-selective nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARxcex3). This receptor and its heterodimeric partner, the retinoid X receptor alpha (RXRxcex1), form a DNA binding complex that regulates transcription of adipocyte-specific genes. Expression and activation of PPARxcex3 in fibroblastic cells triggers the adipocyte gene expression cascade and leads to development of the adipose phenotype.
Accordingly, the development of effective, non-invasive methods for treating liposarcomas would represent a significant advancement in the therapeutic arts.
In accordance with the present invention, we have discovered that PPARxcex3 is expressed consistently in each of the major histologic types of human liposarcoma. It has further been discovered that maximal activation of PPARxcex3 with exogenous ligand (a thiazolidinedione or derivative thereof) promotes terminal differentiation of primary human liposarcoma cells. It has still further been discovered that RXR-specific ligands are also potent adipogenic agents in cells expressing the PPARxcex3/RXRxcex1 heterodimer, and that simultaneous treatment of liposarcoma cells with a thiazolidinedionyl moiety (a PPARxcex3-selective class of compounds) and an RXR-specific ligand results in an additive stimulation of differentiation. Accordingly, according to the invention, there have been identified compositions which are useful for the treatment of liposarcomas.
In accordance with the present invention, there are provided methods for the treatment of liposarcomas, said method comprising administering to a subject in need of such treatment an amount of a therapeutic composition effective to ameliorate the effects of neoplastic cell proliferation of liposarcoma cells, wherein said therapeutic composition comprises at least one thiazolidinedione and at least one retinoid X receptor (RXR) selective agonist in a pharmaceutically acceptable carrier therefor. Invention methods can also be used in a prophylactic manner, i.e., to prevent the onset of liposarcoma.
Thiazolidinediones contemplated for use in the practice of the present invention can be described broadly with reference to the general structure I: 
wherein:
each of X1, X2, X3, X4, X5 and X6 is independently carbon, nitrogen, oxygen or sulfur, with the proviso that at least three of the atoms forming the ring are carbon,
A is:
xe2x80x94Ynxe2x80x94(CRxe2x80x3Rxe2x80x3)mxe2x80x94Z,
xe2x80x94Ynxe2x80x94(CRxe2x80x3Rxe2x80x3)mxe2x80x2xe2x80x94Oxe2x80x94(CRxe2x80x3Rxe2x80x3)mxe2x80x3xe2x80x94Z, or
xe2x80x94Ynxe2x80x94(CRxe2x80x3Rxe2x80x3)mxe2x80x2xe2x80x94N(Rxe2x80x2xe2x80x3)xe2x80x94(CRxe2x80x3Rxe2x80x3)mxe2x80x3xe2x80x94Z,
wherein:
Y is xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94,
n is 0 or 1,
each Rxe2x80x3 is independently hydrogen, lower alkyl, substituted lower alkyl, hydroxy, lower alkoxy, thioalkyl, halogen, trifluoromethyl, cyano, nitro, amino, carboxyl, carbamate, sulfonyl or sulfonamide,
Rxe2x80x2xe2x80x3 is hydrogen, lower alkyl or substituted alkyl,
m falls in the range of 1 up to 15,
each mxe2x80x2 falls independently in the range of 1 up to 8,
each mxe2x80x3 falls independently in the range of 0 up to 12, and
Z is a thiazolidinedionyl moiety;
R2 is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, alkylaryl, substituted alkylaryl, alkenylaryl, substituted alkenylaryl, alkynylaryl, substituted alkynylaryl, arylalkyl, substituted arylalkyl, arylalkenyl, substituted arylalkenyl, arylalkynyl, substituted arylalkynyl, oxyalkyl, poly(alkylene oxide), substituted poly(alkylene oxide); poly(alkylene sulfide), substituted poly(alkylene sulfide), poly(alkylene amine) or substituted poly(alkylene amine); with R2 having in the range of 1 up to about 15 carbon atoms being preferred;
R3 is hydrogen, hydroxy, halogen, alkoxy, lower alkyl, substituted lower alkyl, alkenyl, substituted alkenyl, alkynyl or substituted alkynyl; with R3 having in the range of 0 up to about 6 carbon atoms being preferred;
R4 is hydrogen, formyl, acyl, lower alkyl, substituted lower alkyl or a thiazolidinedionyl moiety; with R4 having in the range of 0 up to about 4 carbon atoms being preferred;
R5 is hydrogen, hydroxy, lower alkoxy, lower alkyl, substituted lower alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl or halogen; with R5 having in the range of 0 up to about 6 carbon atoms being preferred; and
R6 is hydrogen, hydroxy, lower alkoxy, lower alkyl, substituted lower alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl or halogen; with R6 having in the range of 0 up to about 6 carbon atoms being preferred.
Those of skill in the art recognize that the core ring of structure I can be any one of a number of different aromatic or pseudo-aromatic structures, e.g., a benzene ring, a pyridine ring, a pyrazine, an oxazine, and the like.
As employed herein, xe2x80x9clower alkylxe2x80x9d refers to straight or branched chain alkyl groups having in the range of about 1 up to 4 carbon atoms; xe2x80x9calkylxe2x80x9d refers to straight or branched chain alkyl groups having in the range of about 1 up to 12 carbon atoms; xe2x80x9csubstituted alkylxe2x80x9d refers to alkyl groups further bearing one or more substituents such as hydroxy, alkoxy (of a lower alkyl group), mercapto (of a lower alkyl group), halogen, trifluoromethyl, cyano, nitro, amino, carboxyl, carbamate, sulfonyl, sulfonamide, heteroatom-containing cyclic moieties, substituted heteroatom-containing cyclic moieties, and the like.
As employed herein, xe2x80x9calkenylxe2x80x9d refers to straight or branched chain hydrocarbyl groups having at least one carbon-carbon double bond, and having in the range of about 2 up to 12 carbon atoms and xe2x80x9csubstituted alkenylxe2x80x9d refers to alkenyl groups further bearing one or more substituents as set forth above.
As employed herein, xe2x80x9calkynylxe2x80x9d refers to straight or branched chain hydrocarbyl groups having at least one carbon-carbon triple bond, and having in the range of about 2 up to 12 carbon atoms, and xe2x80x9csubstituted alkynylxe2x80x9d refers to alkynyl groups further bearing one or more substituents as set forth above.
As employed herein, xe2x80x9carylxe2x80x9d refers to aromatic groups having in the range of 6 up to 14 carbon atoms and xe2x80x9csubstituted arylxe2x80x9d refers to aryl groups further bearing one or more substituents as set forth above.
As employed herein, xe2x80x9calkylarylxe2x80x9d refers to alkyl-substituted aryl groups and xe2x80x9csubstituted alkylarylxe2x80x9d refers to alkylaryl groups further bearing one or more substituents as set forth above.
As employed herein, xe2x80x9calkenylarylxe2x80x9d refers to alkenyl-substituted aryl groups and xe2x80x9csubstituted alkenylarylxe2x80x9d refers to alkenylaryl groups further bearing one or more substituents as set forth above.
As employed herein, xe2x80x9calkynylarylxe2x80x9d refers to alkynyl-substituted aryl groups and xe2x80x9csubstituted alkynylarylxe2x80x9d refers to alkynylaryl groups further bearing one or more substituents as set forth above.
As employed herein, xe2x80x9carylalkylxe2x80x9d refers to aryl-substituted alkyl groups and xe2x80x9csubstituted arylalkylxe2x80x9d refers to arylalkyl groups further bearing one or more substituents as set forth above.
As employed herein, xe2x80x9carylalkenylxe2x80x9d refers to aryl-substituted alkenyl groups and xe2x80x9csubstituted arylalkenylxe2x80x9d refers to arylalkenyl groups further bearing one or more substituents as set forth above.
As employed herein, xe2x80x9carylalkynylxe2x80x9d refers to aryl-substituted alkynyl groups and xe2x80x9csubstituted arylalkynylxe2x80x9d refers to arylalkynyl groups further bearing one or more substituents as set forth above.
As employed herein, xe2x80x9cpoly(alkylene oxide)xe2x80x9d refers to compounds having the general structure:
xe2x80x94[(CRxe2x80x22)xxe2x80x94O]yxe2x80x94H,
wherein each Rxe2x80x2 is independently selected from hydrogen or lower alkyl, x falls in the range of 1 up to about 4 and y falls in the range of 2 up to about 8; xe2x80x9csubstituted poly(alkylene oxide)xe2x80x9d refers to poly(alkylene oxide) groups further bearing one or more substituents as set forth above.
As employed herein, xe2x80x9cpoly(alkylene sulfide)xe2x80x9d refers to compounds having the general structure:
xe2x80x94[(CRxe2x80x22)xxe2x80x94S]yxe2x80x94H,
wherein Rxe2x80x2, x and y are as defined above; xe2x80x9csubstituted poly(alkylene sulfide)xe2x80x9d refers to poly(alkylene sulfide) groups further bearing one or more substituents as set forth above.
As employed herein, xe2x80x9cpoly(alkylene amine)xe2x80x9d refers to compounds having the general structure:
xe2x80x94[(CRxe2x80x22)xxe2x80x94N(Rxe2x80x2)]yxe2x80x94H,
wherein Rxe2x80x2, x and y are as defined above; xe2x80x9csubstituted poly(alkylene amine)xe2x80x9d refers to poly(alkylene amine) groups further bearing one or more substituents as set forth above.
As employed herein, xe2x80x9cacylxe2x80x9d refers to alkyl-carbonyl species.
As employed herein, xe2x80x9chalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d refers to fluoro substituents, chloro substituents, bromo substituents or iodo substituents.
In a presently preferred aspect of the present invention, xe2x80x9cAxe2x80x9d of Formula I has the structure:
xe2x80x94Ynxe2x80x94(CH2)xxe2x80x94Z
wherein:
Y is xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94,
n is 0 or 1,
x falls in the range of 2 up to 12; and
Z is a thiazolidinedionyl moiety.
In another preferred aspect of the present invention, xe2x80x9cR2xe2x80x9d of Formula I is methyl, ethyl, propyl, butyl, methoxy, ethoxy, propoxy, butoxy, and the like.
In yet another preferred aspect of the present invention, xe2x80x9cR3xe2x80x9d of Formula I is hydrogen, hydroxy, alkoxy, and the like.
In still another preferred aspect of the present invention, xe2x80x9cR4xe2x80x9d of Formula I is selected from formyl, acyl, a thiazolidenedionyl moiety, and the like.
In a further preferred aspect of the present invention, xe2x80x9cR5xe2x80x9d of Formula I is hydrogen.
In a still further preferred aspect of the present invention, xe2x80x9cR6xe2x80x9d of Formula I is hydrogen.
In yet another preferred aspect of the present invention, at least one of R2, R3, R4, R5 and R6 (in addition to A) is not hydrogen. It is especially preferred that at least two of R2, R3, R4, R5 and R6 (in addition to A) are not hydrogen. A plurality of substituents on the ring of structure I is especially preferred when m or the sum of (mxe2x80x2+mxe2x80x3), with reference to the backbone of A, is less than or equal to 6.
Presently preferred species contemplated for use in the practice of the present invention include compounds wherein:
A is a thiazolidinedionyl moiety (e.g., xe2x80x94Oxe2x80x94(CH2)yxe2x80x94thiazolidenedione, wherein y falls in the range of about 2 up to 8),
R2 is hydrogen or lower alkyl,
R3 is hydroxy or alkoxy,
R4 is acyl or a thiazolidenedionyl moiety; and
R5 and R6 are each hydrogen.
The above-described compounds can be readily prepared using a variety of synthetic methods, as are well known by those of skill in the art. For example, many of the above-described compounds can be prepared chemically or enzymatically.
RXR selective ligands contemplated for use in the practice of the present invention include substituted benzoic acids or derivatives thereof (e.g., substituted benzoates), substituted nicotinic acids or derivatives thereof (e.g., substituted nicotinates), substituted carboxylated furans, and the like. Exemplary agonists contemplated for use herein include 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl]benzoic acid, 1,3-propylene glycol ketal of 4-[1-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl]benzoic acid, methyl 4-[(3,8,8-trimethyl-5,6,7,8-tetrahydronaphthalen-2-yl)carbonyl]benzoate, methyl 4-[(3,5,5-trimethyl-5,6,7,8-tetrahydronaphthalen-2-yl)carbonyl]benzoate, methyl 4-[(1,1,2,3,3,6-hexamethylindan-5-yl)carbonyl]benzoate, methyl 6-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)carbonyl]nicotinate, methyl 4-[1-(3,8,8-trimethyl-5,6,7,8-tetrahydro-2-naphthalen-2-yl)ethenyl]benzoate, methyl 4-[1-(3,5,5-trimethyl-5,6,7,8-tetrahydronaphthalen-2-yl)ethenyl]benzoate, methyl 4-[1-(1,1,2,3,3,6-hexamethylindan-5-yl)ethenyl]benzoate, methyl 6-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)ethenyl]nicotinate, 4-[1-(3,8,8-trimethyl-5,6,7,8-tetrahydronaphthalen-2-yl)ethenyl]benzoic acid, 4-[1-(3,5,5-trimethyl-5,6,7,8-tetrahydro-2-naphthalen-2-yl)ethenyl]benzoic acid, 4-[1-(1,1,2,3,3,6-hexamethylindan-5-yl)ethenyl]benzoic acid, 6-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)ethenyl]nicotinic acid, methyl 4-[1-methyl-1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)ethyl]benzoate, 4-[2-methyl-1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)ethyl]benzoic acid, 4-[1-methyl-1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)ethyl]benzoic acid, 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)ethyl]benzoic acid, methyl 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopropyl]benzoate, methyl 4-[2-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)oxiranyl]benzoate, methyl 6-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopropyl]nicotinate, 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopropyl]benzoic acid, 4-[2-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)oxiranyl]benzoic acid, 6-[1-(8 pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopropyl]nicotinic acid (also referred to in the art as xe2x80x9cLG268xe2x80x9d), 3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-ol, methyl 4-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)methyl]benzoate, methyl 4-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)oxy]benzoate, 4-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)methyl]benzoic acid, 4-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)oxy]benzoic acid, methyl 2-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)carbonyl]benzoate, methyl 3-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)carbonyl]benzoate, 2-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)carbonyl]benzoic acid, 3-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)carbonyl]benzoic acid, the carboxylated furan derivative referred to as AGN191701 (see Mol. and Cell. Biol. 15:3540-3551 (1995)), and the like.
Presently preferred RXR selective agonists contemplated for use herein include 6-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopropyl]nicotinic acid and 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)ethenyl]benzoic acid.
Combinations of thiazolidenediones with RXR selective agonists contemplated for use in the practice of the present invention can be employed for both in vitro and in vivo applications. For in vivo applications, the above-described compositions can be incorporated into a pharmaceutically acceptable formulation for administration. Those of skill in the art can readily determine suitable dosage levels when compositions contemplated for use in the practice of the present invention are so used.
In accordance with another embodiment of the present invention, there are provided compositions comprising at least one thiazolidenedione and at least one retinoid X receptor (RXR) selective agonist, wherein said thiazolidenedione has the structure I, as described hereinabove.
In accordance with a particular embodiment of the present invention, compositions comprising at least one thiazolidenedione, an agonist of RXR, and a pharmaceutically acceptable carrier are also contemplated. Exemplary pharmaceutically acceptable carriers include carriers suitable for oral, intravenous, subcutaneous, intramuscular, intracutaneous, and the like administration. Administration in the form of creams, lotions, tablets, dispersible powders, granules, syrups, elixirs, sterile aqueous or non-aqueous solutions, suspensions or emulsions, and the like, is contemplated.
For the preparation of oral liquids, suitable carriers include emulsions, solutions, suspensions, syrups, and the like, optionally containing additives such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents, and the like.
For the preparation of fluids for parenteral administration, suitable carriers include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized, for example, by filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured in the form of sterile water, or some other sterile injectable medium immediately before use.
PPARxcex3 is expressed at high levels in the adipose tissues of mouse and rat (see, for example, Tontonoz et al., in Genes Dev. 8:1224-1234 (1994) and Braissant et al., in Endocrinology 137:354-366(1996)). To determine the tissue distribution of this receptor in humans, Northern analysis of RNA prepared from a variety of human tissues was performed. Human PPARxcex3 is seen to be expressed at highest levels in adipose tissue and at lower levels in several other tissues including lung and kidney. As a control, the blot was also hybridized with cDNA for the adipocyte- specific binding protein aP2. The heart and muscle samples can be seen to contain small amounts of aP2 mRNA, suggesting that these tissues also contain some adipose cells.
Tumorigenesis frequently involves the inactivation or downregulation of genes responsible for initiating and maintaining a differentiated phenotype. As PPARxcex3 appears to play a central role in the adipocyte differentiation process, the expression of this receptor was examined in a series of human liposarcomas. This series included RNA prepared from each of the three major histologic subtypes of liposarcoma; well differentiated/dedifferentiated, myxoid/round cell and pleomorphic. The histologic and cytogenetic characteristics of each tumor is given in Table 3 (see Example 2). For the most part, the well differentiated/dedifferentiated tumors exhibited ring chromosomes and giant marker chromosomes, the myxoid/round cell liposarcomas exhibited the characteristic t(12;16) (13pll) translocation, and the pleomorphic forms exhibited complex rearrangements. Surprisingly, despite their block in differentiation, each liposarcoma examined was found to express high levels of PPARxcex3 RNA, comparable to that of normal fat. These results suggest that most if not all liposarcomas have been transformed at a point in the differentiation process after induction of PPARxcex3 expression. In contrast, PPARxcex3 RNA was not expressed at significant levels in any other type of soft tissue sarcoma examined, including leiomyosarcoma (n=4), fibrosarcoma (n=1), angiosarcoma (n=1), malignant peripheral nerve sheath tumor (MPNS, n=1), or malignant fibrous histiocytoma (MFH, n=1). Thus, PPARxcex3 may be a sensitive marker for distinguishing liposarcoma from other histologic types of soft tissue sarcoma.
Transient transfection experiments were performed to characterize the activation profile of human PPARxcex3. To eliminate interference from endogenous receptor in the transfected cells, a chimeric PPARxcex3 receptor was utilized that could activate transcription through a heterologous response element (see Forman et al., in Cell 83:803-812 (1995)). This chimeric protein contained the yeast GAL4 DNA binding domain linked to the ligand binding domain of human PPARxcex3. The GAL4-hPPARxcex3 expression vector was cotransfected into CV-1 cells with a luciferase reporter plasmid containing the GAL4 upstream activating sequence. In this assay, the thiazolidinediones BRL49653, troglitazone and pioglitazone are all seen to be effective activators of human PPARxcex3.
Liposarcomas have presumably acquired one or more genetic defects that interfere with the course of normal adipocyte development. The observation that PPARxcex3 is expressed consistently in these tumors raised the possibility that the malignant cells might be forced to complete the differentiation program by maximally activating the PPARxcex3 pathway. To address this possibility, primary cells isolated from three human liposarcomas were cultured in vitro. Primary cell strains LS857 and LS175 were derived from well differentiated liposarcomas and LS707 was derived from an intermediate grade myxoid/round cell liposarcoma (see Table 3). High grade pleomorphic liposarcoma cells could not be expanded to sufficient numbers to permit studies of differentiation. A primary leiomyosarcoma cell line LM203 was cultured as a control. To confirm that these cultures consisted of malignant tumor-derived cells, cytogenetic analysis was performed. As shown in Table 3, the karyotype of the cells in each culture was characteristic of the parent liposarcoma.
When cultured in the presence of fetal bovine serum and insulin, conditions permissive for adipocyte differentiation, all three cell lines maintain a fibroblastic morphology. LS175 cells contained small amounts of stainable lipid under these conditions. When cultures were treated for 7 days with 10 xcexcM of the PPARxcex3 ligand pioglitazone, the cells readily accumulated lipid and adopted a morphology characteristic of mature culture adipocytes. No lipid accumulation was observed with the LM203 leiomyosarcoma cells, which do not express PPARxcex3. The degree of morphologically recognizable differentiation varied from 40% in the LS857 cells to 75% in the LS175 cells. After induction for 7 days with thiazolidinedione, cells maintained their differentiated morphology even when pioglitazone was withdrawn. This experiment was performed at least twice with each cell strain with quantitatively and qualitatively similar results. Induction of differentiation was also observed with the thiazolidinediones BRL49653 and troglitazone, while no effect was observed with compound 66, the inactive synthetic precursor to BRL49653.
Simultaneous exposure of competent cells to both PPARxcex3 and RXR-specific ligand was investigated to determine if such combination might provide a stronger adipogenic signal than a PPARxcex3 ligand alone. The ability of the. RXR-specific ligand LG268 to promote adipocyte differentiation was investigated using NIH-3T3 fibroblasts that express PPARxcex3 from a retroviral vector (see Tontonoz et al, in Cell 79:1147-1156 (1994)). It has previously been shown that wild-type NIH-3T3 cells express RXRxcex1, but not PPARxcex3. NIH-vector and NIH-PPARxcex3 cells were cultured as described in the Examples. At confluence, cells were treated for 7 days with no activator, 1 xcexcM pioglitazone alone, 50 nM LG268 alone, or 5 xcexcM thiaozolidinedione and 50 nM LG268. After an additional four days of culture, cells were fixed and stained with oil red O. Data are presented in Table 1 as the range of morphologically recognizable differentiation observed for each line over three separate experiments.
As shown in Table 1, treatment of confluent NIH-PPARxcex3 cells for 7 days with 50 nM LG268 resulted in significant stimulation of adipocyte differentiation, comparable to that seen with 7 days of treatment with 1 xcexcM pioglitazone alone. Thus simultaneous exposure to both activators resulted in an additive effect. LG268 had no effect on NIH-vector cells, indicating that the adipogenic activity of this compound, like that of pioglitazone, is dependent on the presence of PPARxcex3. Similar results are obtained with the preadipocyte cell lines 3T3-L1 and 3T3-F442A, which express both PPARxcex3 and RXRxcex2. Northern analysis confirms that pioglitazone and LG268 have an additive effect on the induction of the adipocyte-specific genes aP2 and adipsin in NIH-PPARxcex3 cells. No induction of adipocyte gene expression was observed in NIH-vector cells under similar conditions.
The ability of LG268 to promote differentiation of human liposarcoma cells was then examined. Treatment of LS857 cells for 7 days with 50 nM LG268 led to a significant degree of adipocyte differentiation, similar to that seen with 10 xcexcM pioglitazone alone. When LS857 cells were treated simultaneously with LG268 and a thiazolidinedione (either pioglitazone or BRL449653), an additive effect on differentiation was observed. To further characterize the effects of PPARxcex3 and RXR ligands on liposarcoma cells, the expression of adipocyte-specific markers were examined by Northern blotting. LS857 cells, like the tumor from which they were derived, express PPARxcex3 mRNA. Treatment of LS857 cells with pioglitazone leads to the induction of two markers of terminal adipocyte differentiation, the MRNAs encoding aP2 and adipsin. Simultaneous treatment with pioglitazone and LG268 results in an additive induction of adipocyte gene expression. In summary, treatment of LS857 cells with thiazolidinediones and RXR-specific retinoids leads to changes in morphology and gene expression consistent with terminal adipocyte differentiation.
Terminal differentiation of white adipocytes in vitro and in vivo is characterized by permanent withdrawal from the cell cycle. A critical question is whether thiazolidinedione-induced differentiation of liposarcoma cells is accompanied by growth arrest. To address this issue, LS857 cells were cultured in the presence or absence of pioglitazone. Following induction of morphologic differentiation, pioglitazone was withdrawn. After 48 hours of continued culture in the absence of pioglitazone, cells were labeled for 48 hours with 5-bromo-2xe2x80x2-deoxyuridine (BrdU). Cells undergoing DNA synthesis during the labeling period should stain positive for BrdU incorporation after fixation and incubation with an enzyme-linked monoclonal antibody (see Examples).
LS857 cells were cultured in the presence or absence of pioglitazone. Following induction of morphologic differentiation, pioglitazone was withdrawn. After 48 hours of continued culture in the absence of pioglitazone, cells were labeled for 48 hours with bromodeoxyuridine, stained using an enzyme-linked monoclonal antibody (see Examples) and visualized microscopically. The number of cells staining positive for BrdU and/or containing visible cytoplasmic lipid is indicated in Table 2.
In the experiments shown in Table 2 (PIO/LG268#1 and PIO/LG268#2), 26-34% of the cells contained visible cytoplasmic lipid. 40-51% of the cells in this culture stained positive for BrdU incorporation by light microscopy; however, of those cells containing lipid, only 3-4% stain positive for BrdU. When differentiated cultures were trypsinized and replated, lipid-containing cells failed to reenter the cell cycle as determined by BrdU labeling. These results demonstrate the thiazolidinedione-induced differentiation of LS857 cells leads to cell cycle withdrawal.
Termination differentiation of most specialized cell types, including white adipocytes, is linked to cell cycle withdrawal. Tumorigenesis is characterized by a loss of cell cycle control and a concordant block in the differentiation program. It has been demonstrated herein that most human liposarcomas express high levels of the adipocyte regulatory complex PPARxcex3/RXRxcex1 and that PPARxcex3 and RXRxcex1-specific ligands are able to trigger terminal differentiation of primary human liposarcoma cells in vitro. These results suggest that the developmental defect in most liposarcomas is downstream of PPARxcex3 expression, and that in at least some tumor cells this developmental block can be overcome by maximal activation of the PPARxcex3 pathway.
While the precise nature of the developmental defects in liposarcoma is not yet clear, it is likely these defects ultimately lead to the inactivation or antagonism of one or more adipocyte transcriptional regulatory proteins. Members of both the C/EBP and PPAR transcription factor families haven been shown to play central complementary roles in adipogenesis in murine models (see, for example, Cornelius et al., in Ann. Rev. Nutr. 14:771-774 (1994), Tontonoz et al., in Curr. Opin. Genet. Dev. 5:571-576 (995), Freytag et al., in Genes Dev. 8:1654-1663 (1994), Wu et al., in Genes Dev. 9: 2350-2363 (1995) and Yeh et al., in Genes Dev 9:168-181 (1995)). Interestingly, the C/EBP family has previously been implicated in the pathogenesis of human myxoid liposarcoma through the characterization of the t(12:16) translocation associated with this tumor. This rearrangement fuses the gene for the C/EBP family member CHOP on chromosome 12 to that of the RNA binding protein TLS on chromosome 16 (see Crozat et al., in Nature 363:640-644 (1993)). CHOP lacks a transcriptional activation domain and has therefore been postulated to function as a dominant negative regulator of other C/EBP proteins. The precise mechanism whereby TLS/CHOP contributes to differentiation arrest and tumorigenesis, however, remains to be elucidated.
The mechanisms by which differentiation is coupled to cessation of cell growth are not fully understood; however, there is mounting evidence that key proteins controlling differentiation interact directly with the cell cycle machinery. For example, the myogenic transcription factor MyoD has been shown to be negatively regulated by cyclin D1-dependent kinase and to induce expression of the cdk inhibitor p21 (see, for example, Halevy et al., in Science 267:1018-1021 (1995) and Skapek et al., in Science 267:1022-1024 (1995)).
The impact of RXR-specific activators on adipocyte differentiation has not previously been addressed. It is demonstrated herein that RXR-specific retinoids can function as adipogenic regulators through activation of the PPARxcex3/RXRxcex1 heterodimer, and that the adipogenic activity of the heterodimer is maximal when both receptors are bound by their respective ligands. Given that PPARxcex3 is likely to be the biologic receptor mediating the insulin-sensitizing effects of the thiazolidinediones, this observation suggests that RXR-specific ligands may also have insulin-sensitizing activity in vivo. Moreover, the insulin-sensitizing effects of thiazolidinedione ligands for PPARxcex3 might be enhanced by simultaneous administration of an RXR-specific ligand.
The results presented herein have important implications for the pharmacologic management of liposarcoma in humans. Liposarcoma is currently managed with surgery and a judicious combination of chemotherapy and radiotherapy. Despite conventional multimodality therapy, anywhere from 25 to 75% of patients with advanced liposarcoma will die from their disease within 5 years. The present results suggest that a combination of the thiazolidinedione class of antidiabetic drugs and RXR-specific retinoids may be useful as a non-toxic alternative to conventional chemotherapy for the treatment of disseminated or locally advanced liposarcoma. Members of the thiazolidinedione class of drugs have undergone extensive preclinical testing as anti-diabetic agents. Troglitazone is currently is phase three clinical trials in the U.S. and studies have supported its usefulness in NIDDM (see Nolan et al., in N. Engl. J. Med. 331:1188-1193 (1994)). Other thiazolidinediones have been approved for clinical use in Japan. Although certain thiazolidinediones have been associated with some degree of toxicity in long-term use as insulin sensitizing agents, this should not preclude their use as antineoplastic agents as conventional chemotherapy is associated with far greater toxicity. The ability of a combination of thiazolidinediones and RXR-specific retinoids to induce differentiation of liposarcoma cells in vitro strongly suggests that these compounds may also be able to stimulate differentiation and growth arrest of human tumors in vivo.